To prepare cytidine diphosphate ribitol (CDP-ribitol) and cytidine diphosphate ribose (CDP-ribose), 2 μM His8-hISPD was incubated at 37°C with either 1 mM ribitol-5-phosphate or 1 mM ribose-5-phoshate as the acceptor substrate in a reaction containing 50 mM Tris-HCl pH 7.4, 1 mM MgCl2, 1 mM DTT, and 1 mM CTP. Reactions were allowed to incubate for 16–18 hr and were stopped by removal of His8-hISPD by filter centrifugation through a Microcon-10kDa Centrifugal Filter Unit with Ultracel-10 membrane (EMD Millipore). Reaction products were confirmed using a linear ion trap-Fourier transform mass spectrometer (LTQ-Orbitrap Discovery, Thermo-Fisher, San Jose, CA). Reaction products were mixed with an equal volume of 80% acetonitrile and 0.1% formic acid and analyzed by direct infusion in negative ion mode using a nanospray ion source with a fused-silica emitter (360 × 75 × 30 μm, SilicaTip, New Objective) at 1.5 kV capillary voltage, 200°C capillary temperature, and a syringe flow rate of 1 μL/min. All products were confirmed by MS/MS ion trap mass spectrometry (ITMS) acquired at 45% collision-induced dissociation (CID) and 2 m/z isolation width.
Microcon 10kda centrifugal filter unit with ultracel 10 membrane
Manufactured by Merck Group
Sourced in Italy, Ireland
The Microcon-10kDa Centrifugal Filter Unit with Ultracel-10 membrane is a lab equipment product manufactured by Merck Group. It is designed to concentrate and desalt macromolecular solutions using centrifugation. The unit contains an Ultracel-10 regenerated cellulose membrane with a molecular weight cutoff of 10 kDa.
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Lab products found in correlation
Lysosome isolation kit, by Abcam (2 mentions)
Ltq orbitrap discovery, by Thermo Fisher Scientific (1 mentions)
Silicatip, by New Objective (1 mentions)
Formic acid, by Merck Group (1 mentions)
Nitrate nitrite fluorometric assay kit, by Cayman Chemical (1 mentions)
Abx pentra 400, by Horiba (1 mentions)
Mem per plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Nitrate nitrite colorimetric assay kit, by Cayman Chemical (1 mentions)
Synergy h1 microplate reader, by Agilent Technologies (1 mentions)
Microtainer blood collection tube, by BD (1 mentions)
8 protocols using microcon 10kda centrifugal filter unit with ultracel 10 membrane
1
Enzymatic Synthesis of CDP-Ribitol and CDP-Ribose
2
Aptamer-Antibiotic Binding Analysis
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Vials with 100 µL solutions of macromolecules:antibiotic (1:1 volumes) were prepared at a fixed antibiotic concentration and increasing macromolecule concentration and left to reach equilibrium for 30 min. Afterwards, the solutions were transferred in the upper compartment of filters with a nominal cutoff of 10.000 Dalton (Microcon-10kDa Centrifugal Filter Unit with Ultracel-10 membrane, Merck Life Science S.r.l.; Milan, Italy), or 3.000 Dalton (Amicon Ultra-0.5 Centrifugal Filter Unit, Ultracel-3, Merck Life Science S.r.l.; Milan, Italy), depending on the MW of the tested aptamer. Then, the solutions were centrifuged for 8 min at 12.000× g, allowing around 50–60 µL of solution to flow to the lower filter compartment. The antibiotic concentration found in the flow through is a good estimate of the free antibiotic present in the original reaction vial. On the other hand, it is expected that the antibiotic bound to the aptamer is (mostly, see below for details) confined in the upper filter compartment.
3
Optimized LC-MS Sample Preparation
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All operations were performed using chemical reagents of optima LC–MS grade. Solvents and formic acid were obtained from Sigma-Aldrich (Poole, Dorset, UK) or Thermo Fisher Scientific (Loughborough, Leicester, UK). Total recovery LC–MS vials were purchased from Waters (Elstree, Hertfordshire, UK). Microcon-10 kDa Centrifugal Filter Unit with Ultracel-10 membrane (Merck Millipore Ltd., Cork, Ireland) and Corning Costar Spin-X centrifuge tube filters with cellulose acetate membranes of 0.22 µm (Corning, Inc, USA) were purchased from Merck.
4
Rat Plasma Biomarker Profiling
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Blood samples were collected in K2‐EDTA tubes from the caudal vein under 1.5–2% isoflurane anaesthesia at baseline and after 5 weeks of treatment. Blood lymphocyte count was measured by MS9–5 (Melet Schloesing) in freshly collected blood, then blood samples were immediately centrifuged at 4°C/10 min/10 000 g and the plasma was stored at −80°C. Plasma creatinine and fructosamine were measured by ABX PENTRA 400 (Horiba). SAR247799 concentrations were measured from 5‐week plasma samples using liquid chromatography tandem mass spectrometry.
Plasma biomarkers were measured by ELISA following 5 weeks of treatment according to manufacturers' instructions. Soluble ICAM‐1, sE‐selectin and von Willebrand Factor (vWF) were measured using MILLIPLEX MAP Rat Vascular Injury Magnetic Bead Panel 2 (Merck), sVCAM‐1 from VCAM‐1 Rat ProcartaPlex Simplex Kit (ThermoFisher Scientific), CRP from CRP Rat ProcartaPlex Simplex Kit (ThermoFisher Scientific) and sPECAM‐1 from Rat PECAM‐1 Elisa kit (MyoBioSource). Nitrate/nitrite concentration was measured by nitrate/nitrite fluorometric assay kit (Cayman Chemical) in plasma samples previously filtered with Microcon‐10 kDa Centrifugal Filter Unit with Ultracel‐10 membrane (Merck).
Plasma biomarkers were measured by ELISA following 5 weeks of treatment according to manufacturers' instructions. Soluble ICAM‐1, sE‐selectin and von Willebrand Factor (vWF) were measured using MILLIPLEX MAP Rat Vascular Injury Magnetic Bead Panel 2 (Merck), sVCAM‐1 from VCAM‐1 Rat ProcartaPlex Simplex Kit (ThermoFisher Scientific), CRP from CRP Rat ProcartaPlex Simplex Kit (ThermoFisher Scientific) and sPECAM‐1 from Rat PECAM‐1 Elisa kit (MyoBioSource). Nitrate/nitrite concentration was measured by nitrate/nitrite fluorometric assay kit (Cayman Chemical) in plasma samples previously filtered with Microcon‐10 kDa Centrifugal Filter Unit with Ultracel‐10 membrane (Merck).
5
Mitochondrial Protein Detection Protocol
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Mitochondrial fractions were isolated to detect mitochondrial proteins using a mitochondria isolation kit for cell cultures (Catalog: 89874, Option B method) from Life Technologies, Thermo Fisher Scientific, according to the manufacturer's instructions. The cytosolic fraction from the mitochondrial preparation was further concentrated using a Microcon-10 kDa Centrifugal Filter Unit with Ultracel-10 membrane (Millipore) to detect cytosolic proteins. Protein was estimated by the Bradford assay and 20–30 µg of protein were used for western blotting. The purity of the mitochondrial fractions was established by the absence of cytosolic or nuclear proteins, LDH and Lamin B1, respectively, as described before (Devi et al., 2017 (link)).
6
Lysosomal Membrane Protein Extraction
7
Serum Nitrate/Nitrite Analysis in Mice
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Blood from indicated mouse strains was harvested via heart puncture and centrifuged at 10,000 × g for 6 minutes in serum separation tubes (BD Microtainer Blood Collection Tubes Cat. No. 365967). Serum was filtered through a Microcon-10kDa Centrifugal Filter Unit with Ultracel-10 membrane (Millipore Cat. No. MRCPRT010) per product manual. Filtered serum was diluted 1:8 in assay buffer and measured for total nitrate and nitrite using Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical Cat. No. 780001). Absorbance was measured at 540 nm using a BioTek Synergy H1 Microplate Reader, and total nitrate and nitrite calculated from absorbance according to product manual.
8
Lysosomal Fraction Isolation Protocol
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Supernatants of cells were concentrated using a Microcon-10kDa Centrifugal Filter Unit with Ultracel-10 membrane (Millipore, MRCPRT010). The lysosome isolation kit (Abcam, ab234047) was used to isolate lysosomal fractions from cultured cells by differential centrifugation followed by density gradient centrifugation. Finally, the purified lysosomal fraction was obtained using an ultracentrifuge (145,000 × g) for 2 h at 4°C.
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